In immune-mediated inflammatory diseases, numerous cell types of the immune system, such as neutrophils, monocytes, macrophages, eosinophiles, mast cells and lymphocytes play often a major causative role. The active recruitment and accumulation of these cells are life-saving in some circumstances (e.g., bacterial or nematode infection) but life-threatening in others (e.g., allergy). In particular, mast cells have been considered for many years to participate specifically in allergic reactions through the release of cytokines, chemokines, proteases, leukotrienes, and bioactive polyamines. Emerging roles for mast cells have been identified recently, which highlight their relevance in both innate and adaptive immunity, as well as pathological inflammatory conditions (Shea-Donohue T. et al. (2010), Curr Gastroenterol Rep., 12(5) p. 349-357).
The serine protease chymase (EC=3.4.21.39) is a chymotrypsin-like enzyme that is expressed in the secretory granule of mast cells (Miller H. and Pemberton A., (2002), Immunity, 105, p. 375-390). Upon release, it has been described to degrade the extracellular matrix (e.g., proteoglycans, collagen, elastin, fibronectin), induce leukocyte migration and cytokine production, activate TGF-beta and MMP-9, and promote tissue remodeling (de Garavilla L. et al. (2005) J Biol Chem, 280(18) p. 18001-18007 and Takai S. et al. (2010) J Pharmacol Sci, 113(4), p. 301-309). In addition to these pro-inflammatory effects upon release from the granules of mast cells in different types of tissues, chymase has been found in mast cells in the human heart, where it cleaves angiotensin I to form angiotensin II (Urata et al. (1993) J Clin Invest, 91(4), p. 1269-1281). Angiotensin II in the blood has multiple diverse physiological effects on the cardiovascular system, including arteriolar vasoconstriction and aldosterone secretion. In the heart itself, it has positive inotropic and chronotropic effects (Peach et al. (1977), Physiol. Rev., 57, p. 313-370). In cardiovascular pathologies (such as hypertension), blockade of angiotensin II mediated effects by angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor blockers has shown the usefulness of treating this diseases, as demonstrated by the large number of different marketed compounds (Lüllmann H. and Mohr K., in: Taschenatlas der Pharmakologie, 4th edition, Georg Thieme Verlag Stuttgart/New York, 2001, p. 126-127).
Because of the pro-inflammatory enzymatic activities of chymase, and because chymase generates angiotensin II by an ACE-independent pathway in the heart, it has been postulated that inhibition of chymase promises new therapeutic approaches to prevent organ and tissue damage in inflammatory conditions and cardiovascular diseases. According to this rationale, chymase inhibitors have been generated and were investigated as anti-inflammatory and cardioprotective agents in a number of in vivo studies, demonstrating that chymase inhibition reduces inflammatory processes: For example, in initial studies, the inhibition of chymase in an acute peritonitis model in rats reduced the up-regulation of pro-inflammatory cytokines in plasma and/or peritoneum and reduced the influx of neutrophils into the peritoneal cavity (de Garavilla L. et al. (2005) J Biol Chem, 280(18) p. 18001-18007). On the basis of this success, it was investigated whether chymase inhibition is relevant for the treatment of asthma and chronic obstructive pulmonary disease (COPD). Three different animal models of inflammation with pathological responses related to those manifested in allergic asthma and/or COPD were investigated (Maryanoff B. et al. (2010), Am J Respir Crit Care Med, 181 p. 247-253): (1) mast-cell mediated inflammation in ovalbumin sensitized rats, (2) allergen-induced bronchoconstriction and airway hyperresponsiveness in allergic sheep, and (3) tobacco smoke induced neutrophilia in mice. The results presented by the authors indicate that the chymase inhibitor is able to disrupt inflammatory sequelae in animal models associated with diseases related to asthma and COPD (Maryanoff B. et al. (2010), Am J Respir Crit Care Med, 181 p. 247-253). Moreover, a chymase inhibitor prevented vascular proliferation after balloon catheter injury in a dog model (Takai S. et al., (2003), J Pharmacol Exp Ther, 304, p. 841-844). In addition, it was shown that hamsters treated with combined ACE and chymase inhibitors, relative to ACE inhibition alone, improved left ventricular function, decreased adverse cardiac remodelling and improved survival after myocardial infarction. Other animal studies demonstrated efficacy of chymase inhibitors in animal models of myocardial infarction, cardiomyopathy, and tachycardia-induced heart failure (Doggrell Sh. and Wanstall J., (2005), Can J Physiol Pharmacol, 83, p. 123-130). Takai et al. teach that chymase inhibitors may be useful to prevent organ damages in a number of diseases, such as: aortic aneurysm, diabetic retinopathy, fibrosis, ulcerative colitis and other inflammatory disorders (Takai S. et al. (2010) J Pharmacol Sci, 113(4), p. 301-309).
In summary, inhibition of chymase appears a useful modality in inflammatory conditions, in which chymase blockade is desired, such as allergy, asthma, COPD, rheumatoid arthritis, ulcerative colitis, diabetes, Crohn's disease, cardiomyopathy, myocardial infarction, left ventricular hypertrophy, unstable angina pectoris, restenosis and atherosclerotic plaques. Only recently Diaconu et al. (2011), Arch Dermato Res., ahead of print described a role of chymase in cancer, in particular in uterine cervical carcinoma.
In general, the development of compounds that inhibit protease targets (proteases comprise more than 500 family members) with high affinity and specificity has proved challenging in the past. This is reflected by the fact that although proteases are considered as an important target class (because unregulated proteolysis leads to many pathologies, as exemplified above for chymase) it is notable, that there are only few marketed drugs (such as HIV protease inhibitors, angiotensin-converting enzyme (ACE) inhibitors, a kallikrein inhibitor, the proteasome inhibitor bortezomib, the recently approved renin-inhibitor aliskiren, anticoagulation drugs and DPP4-inhibitors). The key challenge in the discovery of new protease inhibitors is the ability to identify drugs which are both potent and specific (Drag M. and Salvesen G., (2010), Nat Rev Drug Discov., 9(9), p. 690-701) as structural similarities within the active site of most proteolytic enzyme families often result in a simultaneous inhibition of several family members, which can lead to an unacceptable toxicity profile of the drug.
One avenue of obtaining high affinity and specific inhibitors of chymase with desired pharmacokinetic and pharmacodynamic properties, represents the use of antibody and alternative binding technologies (the latter termed “scaffolds”, see below). Antibodies represent the best established class of binding molecules in the field of pharmaceutical biotechnology. But, even though antibodies are routinely employed for analytical, purification, diagnostic and therapeutic purposes due to their ease of production, high affinity and specificity to virtually any desired target antigen, these still have a number of serious drawbacks such as the necessity of complex mammalian cell production systems, a dependency on disulfide bond for stability, the tendency of some antibody fragments to aggregate, limited solubility and last but not least, they may elicit undesired immune responses even when humanized. As a consequence, a recent focus for developing small globular proteins as scaffolds for the generation of novel classes of versatile binding proteins has emerged. For generating diversity and target specificity, typically surface components (e.g. extracellular loops) of a protein framework with suitable biophysical properties are combinatorially mutated for producing a protein library to be screened for the target binding specificities of interest (Binz, H. K., and Pluckthun, A. (2005) Curr. Opin. Biotechnol. 16, 459-469).
These non-immunoglobulin-derived binding reagents are collectively designated “scaffolds” (Skerra A. (2000) J. Mol. Recognit. 13, 167-187). More than 50 different protein scaffolds have been proposed over the past 10 to 15 years, the most advanced approaches in this field being (as summarized in Gebauer M and Skerra A. (2009) Curr Opinion in Chemical Biology 13:245-255): affibodies (based on the Z-domain of staphylococcal protein A), Kunitz type domains, adnectins (based on the 10th domain of human fibronectin), anticalins (derived from lipocalins), DARPins (derived from ankyrin repeat proteins), avimers (based on multimerized LDLR-A), affitins (based on Sac7d from the hyperthermophilic archaeon), and Fynomers, which are derived from the human Fyn SH3 domain.
In general, SH3 domains are present in a large variety of proteins participating in cellular signal transduction (Musacchio et al. (1994) Prog. Biophys. Mol. Biol. 61; 283-297). These domains do not occupy a fixed position within proteins and can be expressed and purified independently. More than 1000 occurrences of the domain are presently known with about 300 human SH3 domains (Musacchio A. (2003) Advances in Protein Chemistry. 61; 211-268). Although there is great sequence diversity among SH3 domains, they all share a conserved fold: a compact beta barrel formed by two anti-parallel beta-sheets (Musacchio A. (2003) Advances in Protein Chemistry. 61; 211-268). Typically, SH3 domains bind to proline-rich peptides containing a PXXP core-binding motif (Ren et al. (1993) Science 259; 1157-1161), but examples of unconventional SH3 binding sites have also been described (Karkkainen et al. (2006) EMBO Rep. 7; 186-191). Most of the SH3 domains sequenced so far have an overall length of approximately 60 to 65 amino acids, but some of them may feature as many as 85 amino acids due to inserts into the loops connecting the main conservative elements of the secondary structure (Koyama et al. (1993) Cell 72(6); 945-952). An alignment of different SH3 domains revealed conserved amino acid residues responsible for the proper structure formation as well as for the canonical proline-rich motif recognition (Larson et al. (2000) Protein Science 9; 2170-2180).
Recently it was demonstrated that the Fyn SH3 domain is an attractive scaffold (“Fynomer”) for the generation of binding proteins because it (i) can be expressed in bacteria in soluble form in high amounts, (ii) is monomeric and does not aggregate when stored in solution, (iii) is very stable (Tm 70.5° C.), (iv) lacks cysteine residues, and (v) is of human origin featuring an amino acid sequence completely conserved from mouse to man and, hence, non-immunogenic (Grabulovski et al. (2007) JBC, 282, p. 3196-3204; EP 2054432).
Whereas chymase is a therapeutically attractive target, to the best knowledge of the inventors, so far no Fynomer-based binders have been described in the art. It was thus challenging to identify and describe features required for binding molecules to chymase that would also provide an impact on their therapeutic applicability.
Thus, the objective underlying the present invention is to provide new potent chymase binding molecules, in particular ones with high specificity and high affinity for chymase. It is a further objective to provide chymase-binding molecules, preferably chymase inhibitors, suitable for research, diagnostic and medical treatment, preferably for use in medicaments for treating and/or preventing chymase-mediated diseases and medical conditions.